Figure 9.1 Macrophage stained with FITC labeled anti-tubulin
Cell shape is determined by microscaffolding created by microtubules and microfilaments. Internally, these tubules and filaments make up the cytoskeleton. Externally they are the components of the extracellular matrix (glycocalyx and plant cell wall).
Microtubules have a diameter of about 24 nm and are usually
found in groups of 13 protofilaments. The basic structural
subunit is a dimer of two similar proteins, 
and 
tubulin,
each with a molecular weight of 55,000 daltons. Tubulin and
several larger microtubule-associated proteins (MAPs) interact in
the presence of GTP and Mg
to form hollow tubes known as
protofilaments which can stretch for long distances across the
cytoplasm. The MAPs function to anchor the tubulin protofilaments
together into larger structures (centrioles, basal bodies,
flagella) and to attach the protofilaments to other structures
within the cell (nuclear envelope, chromosomes) as well as to
other filamentous molecules (actin, intermediate filaments).
Polymerized tubules are also important in cell division,motility, communication and differentiation (morphogenesis). They are believed to be important elements of neural cell function and glandular secretion.
Within a cell, microtubule integrity is affected by
temperature. Microtubules are relatively stable at physiological
temperatures, but below 10° C they spontaneously
depolymerize into constituent subunits, namely tubulin and the
microtubule-associated proteins (MAP's). When the cells are
returned to 37° C, microtubules are reformed in about 15
minutes (providing that sufficient GTP and Mg are present).
Microtubules
One of the several methods currently used to isolate
microtubules takes advantage of the temperature-dependent
stability of the microtubule proteins. Polymerized microtubules
maintain their structural organization at physiological
temperatures, but rapidly depolymerize below 10° C.
A crude extract of tissue containing large numbers of
microtubules is warmed to 37° C in a buffer
containing GTP, EGTA (a chelating agent that binds calcium ions)
and Mg. Microtubules will form in approximately 15 minutes.
As the proteins polymerize into tubules, the viscosity of the solution will increase. Increased viscosity can be monitored directly with a viscometer, or indirectly by a change in sedimentation coefficient or by an alteration in the turbidity of the solution. Turbidity is the easiest alteration to monitor, since it can be measured with a UV spectrophotometer.
After the formation of the microtubules, a pellet enriched in microtubules may be prepared by centrifugation of the viscous solution at warm temperatures (10° C). The pellet is then depolymerized by incubation at 4° C in buffer, once again yielding a solution of tubulin, with a slight decrease in yield, but an increase in the purity. The supernatant containing both tubulin and microtubule-associated protein is again warmed for microtubule polymerization. Several cycles of this temperature dependent, assembly/disassembly procedure results in highly pure preparations of microtubules.
The polymerized microtubule structure can be examined by transmission electron microscopy for structural integrity. The microtubules are negatively stained and dried directly onto a coated EM grid for observation. Alternatively, they can be pelleted, embedded and sectioned for routine TEM analysis. Since the tubulin and MAPs are protein, antibodies can be made against the molecules. These can be used either for EM analysis or for immunofluorescent analysis at the light microscope level.
Microtubule assembly can be inhibited by several chemical agents. Colchicine or vincristine depolymerize microtubules, and/or prevent their polymerization from the monomer tubulin. Colchicine is an alkaloid extracted from the plant Colchicum sativum. Colcemid, a synthetic derivative, is commonly used in tissue cultures to halt mitotic cells at metaphase. When colcemid is removed, spindle fibers will form and the cell will divide. If the colcemid treatment is prolonged, the cell will not divide and will result in polyploid formation..
Immunofluorescent Analysis
Cells fixed at 37° C in their microtubular structure. To visualize the organization of the microtubules, plasma membranes are solubilized and made permeable to anti-tubulin antibodies. If the antibody has previously been bonded to a fluorescent dye, the organization of microtubules within the cell can be observed with a fluorescent microscope.
Microfilaments
One of the most studied forms of molecular architecture associated with cellular function has been the relationship of thick and thin filaments of the striated muscle of vertebrates. The thin actin filaments (6-8 nm diameter) are interposed between the thick myosin filaments (18 nm diameter) and these in turn are often associated with intermediate filaments (about 10 nm diameter).
Figure 9.1 presents a schematic of the relationship of these filaments in a typical striated muscle while Figure 9.2 gives both light and TEM views.
In smooth muscle, the connections are not as clearly defined, but the presence of the three types of filaments can be observed. In smooth muscle, the intermediate filaments are integrated into the thin filaments.
Imtermediate Filaments
Any analysis of microfilaments would be incomplete without some study of the host of filaments collectively referred to as intermediate filaments.
There are five types of intermediate filaments: tonofilaments (keratins or cytokeratins), neurofilaments, glial filaments, desmin filaments and vimentin filaments. For further details, refer to Table 9.1.
GAGs
There are a host of filamentous and globular proteins on the surface of cells which are composed of both proteins and carbohydrates. Older terminology referred to these as glycoproteins if there was more protein than carbohydrate and mucopolysaccharides if there was more carbohydrate. Today, the common term is Glycos Amino Glycans or GAGs. Examples of GAGs are collagenous and non-collagenous glycoproteins, elastin, sulphated proteoglycans (heparin) and complexes with hyaluronic acid.
GAGs are involved in the formation of the "pericellular" matrices of developing cells. These matrices are important for cell-cell contact in early embryogenesis and differentiation, and in the formation of extracellular basement membranes. There is evidence that they are involved in information processing of RNA within the cell as well as their structural function. Receptor mediated endocytosis is also dependent upon these surface molecules as is normal receptor activities of hormones, neurohormones and the activation of cAMP.
